CAROTINOIDS IN THE PHANEROGAMS 51 



eal methods which they used, it will In- necessary to state brielly the 

 character and significance of I he methods, reserving a fuller descrip- 

 tion t'nr a later chapter. 



Frank (1884) first observed that red crystalline needles form in the 

 plastids and between the chlorophyll granules when green leaves are 

 immersed in dilute acids for a time, and then, after washing off the 

 acid, are allowed to remain in distilled water for a still more pro- 

 tracted period. Tschirch (1884 1, who first examined the phenomenon, 

 did not decide the nature of the crystals, but Molisch 1 1890) found the 

 crystals to be identical in properties, although having a more reddish 

 color, with the majority of crystals which he found could be produced 

 by an entirely different method. Molisch's method is to immerse the 

 leaves in dilute (40 per cent by volume) alcohol containing 20 per 

 cent KOH, until the chlorophyll is completely extracted. The process 

 sometimes requires several days. On washing off the green extract 

 with water, and immersing the washed leaves in distilled water for 

 several hours to insure the complete removal of the chlorophyll, it is 

 found that crystals of various forms and colors from yellowish-orange 

 to red have appeared abundantly in the leaf. Molisch proved fairly 

 conclusively the identity of many of the crystals thus obtained with 

 the red-orange crystals which form in concentrated alcoholic leaf 

 extracts, and accordingly decided to call the crystals carotin. Molisch 

 was care"ful to point out, however, that he used the term carotin in the 

 sense of a group of closely related pigments, for he recognized that the 

 crystals formed by his alkali method were not due in all cases to the 

 same pigment. Tammes (1900) and Kohl (1902), however, who 

 greatly extended our knowledge of the presence of carotinoids in the 

 plant kingdom, using the microchemical methods of Frank and 

 Molisch, believed that only one pigment was concerned, namely, caro- 

 tin, and regarded the methods as specific for this pigment. Tswett 

 1 191 la), however, proved definitely that the crystals obtained by 

 Molisch's method are a mixture of carotinoids, and this has been com- 

 pletely confirmed by van Wisselingh (1915i. Other microchemical 

 methods for carotinoids have been worked out by the two investi- 

 gators just mentioned, and these will be reviewed in. a later chapter. 

 Tswett has stated that Frank's acid method may possibly be specific 

 for carotin. This may well be the case in view of the much greater 

 sensitiveness of the xanthophylls to acids, as van Wisselingh has 

 pointed out, but this investigator who has studied the method closely 

 finds it to be often laborious, requiring sometimes several months for 



